When beginning a new project, it will be necessary to iteratively
optimize samples between the TEM and FIB. For example, the final
lamella thickness should be guided by the intended TEM data
acquisition pixel size. This is important in order to limit the total
dose applied to a specimen during tilt series acquisition. Doubling
the TEM magnification while maintaining a similar beam intensity
on the camera will result in four times the applied dose (in electrons
per square angstrom) to the sample. A higher desired TEM magnification will therefore require thinner lamellae in order to reduce
the dose applied to the specimen while maintaining an appropriate
beam intensity on the camera. Conversely, thick specimens will
require additional radiation, resulting in lower signal-to-noise
ratio and lower resolution, but allow imaging of a larger volume
for increased cellular context.
For reasons described above, it is important to track and orient
grids during FIB milling and through to TEM data acquisition.
This is most easily accomplished by using marked autogrid supports
that allow the user to orient the sample at the aquilos loading
station and at the TEM loading station. Examples of marked autogrids include the commercially available cryo-FIB autogrids that
have laser-etched dots in the surface of the ring to indicate sample
orientation. These marks may be difficult to see under liquid nitrogen, so we routinely mark the autogrids with a colored permanent
marker.
While cryo-FIB milling exhibits fewer artifacts compared to
sections from microtomy, the ion beam will damage the surface of
the lamella as it ablates material [46]. Additionally, repeated use of
the SEM for monitoring milling progress also contributes electron
damage. In particular, sensitive structures such as bacterial polyphosphate bodies, accumulated damage from the FIB/SEM can be
seen as localized bubbling confined to the surface of the specimen
to several nanometers deep. However, previous work has demonstrated that FIB milled samples do not exhibit heat-induced devitrification beyond the immediate interacting surface [46].
3.4 Common
Troubleshooting Items
3.4.1 System
Maintenance
In addition to manufacturer suggested regular instrument upkeep,
we recommend following a regular maintenance schedule for cryoFIB/SEM systems focused on checking stage temperature, cleaning the stage of platinum buildup, and cleaning seals/o-rings of
dust. These tasks should be done every 3–4 months. Additionally,
the stage components should be inspected for wear and defects
regularly due to the large temperature cycles that these parts
experience.
System consumables include the gallium liquid-metal ion-source and the FIB aperture strip, both of which may need replacing every 6 months if the instrument is used routinely.
Practical Approaches for Cryo-FIB Milling
73
optimize samples between the TEM and FIB. For example, the final
lamella thickness should be guided by the intended TEM data
acquisition pixel size. This is important in order to limit the total
dose applied to a specimen during tilt series acquisition. Doubling
the TEM magnification while maintaining a similar beam intensity
on the camera will result in four times the applied dose (in electrons
per square angstrom) to the sample. A higher desired TEM magnification will therefore require thinner lamellae in order to reduce
the dose applied to the specimen while maintaining an appropriate
beam intensity on the camera. Conversely, thick specimens will
require additional radiation, resulting in lower signal-to-noise
ratio and lower resolution, but allow imaging of a larger volume
for increased cellular context.
For reasons described above, it is important to track and orient
grids during FIB milling and through to TEM data acquisition.
This is most easily accomplished by using marked autogrid supports
that allow the user to orient the sample at the aquilos loading
station and at the TEM loading station. Examples of marked autogrids include the commercially available cryo-FIB autogrids that
have laser-etched dots in the surface of the ring to indicate sample
orientation. These marks may be difficult to see under liquid nitrogen, so we routinely mark the autogrids with a colored permanent
marker.
While cryo-FIB milling exhibits fewer artifacts compared to
sections from microtomy, the ion beam will damage the surface of
the lamella as it ablates material [46]. Additionally, repeated use of
the SEM for monitoring milling progress also contributes electron
damage. In particular, sensitive structures such as bacterial polyphosphate bodies, accumulated damage from the FIB/SEM can be
seen as localized bubbling confined to the surface of the specimen
to several nanometers deep. However, previous work has demonstrated that FIB milled samples do not exhibit heat-induced devitrification beyond the immediate interacting surface [46].
3.4 Common
Troubleshooting Items
3.4.1 System
Maintenance
In addition to manufacturer suggested regular instrument upkeep,
we recommend following a regular maintenance schedule for cryoFIB/SEM systems focused on checking stage temperature, cleaning the stage of platinum buildup, and cleaning seals/o-rings of
dust. These tasks should be done every 3–4 months. Additionally,
the stage components should be inspected for wear and defects
regularly due to the large temperature cycles that these parts
experience.
System consumables include the gallium liquid-metal ion-source and the FIB aperture strip, both of which may need replacing every 6 months if the instrument is used routinely.
Practical Approaches for Cryo-FIB Milling
73